Biomaterial-mediated retroviral gene transfer using self-assembled monolayers
Introduction
Genetic engineering of mammalian cells is central to numerous strategies for disease treatment, tissue regeneration, and the study of protein function and cellular processes [1], [2], [3]. Conventional methods for gene delivery include both in vitro and in vivo gene transfer based on viral vectors or DNA-polymer/matrix complexes. In vitro gene transfer typically involves the direct transfection or transduction of cultured cells that are subsequently analyzed for transgene activity and/or implanted for a therapeutic purpose. In vivo gene therapy involves the direct application of the gene carrier to the injured or diseased tissue. However, these approaches are often limited by inefficient transgene delivery and poor specificity. Recent attempts to overcome these limitations have focused on biomaterial-mediated gene delivery, wherein the gene carrier is immobilized to, or encapsulated within, a biomaterial support [1], [2], [3]. The hybrid gene-activated biomaterial is then seeded with cells in vitro or implanted. By co-localizing the gene delivery vehicle and cell adhesion, this method enhances gene transfer and specifically targets cells at the biomaterial interface, thereby reducing the risks associated with direct injection of the gene carrier. Therefore, this approach provides several advantages over conventional gene delivery modalities, including reduced cytotoxicity and immunogenicity of freely diffusible gene carriers, limited ectopic transgene expression in neighboring tissues, improved stability of the gene carrier, and controlled levels of gene transfer and expression.
Strategies for the integration of biomaterials and gene delivery have targeted both viral and plasmid DNA-based gene carriers. Fang et al. originally developed this method in the form of a gene-activated matrix (GAM) in which plasmid DNA was loaded onto collagen sponges prior to implantation into segmental bone defects [4]. Plasmid DNA was later incorporated into poly(lactide-co-glycolide) (PLG) scaffolds with tunable degradation properties, allowing for sustained release of DNA from the scaffold for up to 1 month [5]. Subsequently, numerous studies have evaluated strategies for controlled release of plasmid DNA from polymeric scaffolds or immobilization of DNA at biomaterial surfaces [1]. Methods for plasmid release have primarily focused on DNA incorporation into biodegradable polymers, including collagen [4], [6], hyaluronan [7], [8], PLG [5], [9], poly(ethylene glycol) [10], and ethyl vinyl-co-acetate [11]. Immobilization approaches typically utilize the interaction of DNA with cationic agents, including poly-l-lysine [12], polyethylenimine [13], [14], [15], chitosan [16], or dendrimers such as polyamidoamine [17]. More recently, efforts have focused on incorporating the high gene transfer efficiencies of viral vectors into biomaterial-based gene delivery. For example, adenovirus particles have been suspended in hydrogel microspheres of fibrin [18], collagen [18], alginate [19], and poly(lactic-co-glycolic acid (PLGA) [20], [21] and implanted for controlled delivery in vivo. Additionally, Schwarz and colleagues have developed a technique for freeze-drying recombinant adeno-associated virus particles onto implants to enhance tissue repair [22].
Although many strategies exist for biomaterial-mediated gene therapy with plasmid DNA or adenoviral particles, both of these methods generate only transient transgene expression. There are many examples of biomedical applications that require prolonged or sustained expression of the transgene. Therefore, an unfulfilled need exists for methods that immobilize or release gene carriers that permanently modify the cellular DNA, such as retroviruses and lentiviruses. In contrast to adeno- and adeno-associated viruses, these particles consist of an unstable lipid bilayer and RNA genome that present unique challenges for incorporation into biomaterials. Retrovirus immobilization to human plasma fibronectin or recombinant fibronectin fragments has been widely utilized to enhance gene delivery to several cell types [23], [24], [25]. However, fibronectin is an intrinsically bioreactive molecule that directs confounding effects on cell differentiation and proliferation [26], [27], [28]. Cationic and anionic polymers have been used to form large virus-polymer complexes and increase the rate of virus sedimentation onto cultured cells [29], [30], [31]. Building on these results, other studies have analyzed the effects of cationic surfaces or molecules to regulate the immobilization and delivery of negatively-charged retrovirus particles [32], [33], [34]. The present study expands on this work by evaluating the effects of surface chemistry on biomaterial-mediated gene delivery.
Self-assembled monolayers (SAMs) represent a robust, controlled, and stable biomaterial for analyzing the effects of surface chemistry on biological phenomena [35]. SAMs have been used extensively to study and control protein adsorption and cell adhesion and function in vitro and in vivo. More recently, plasmid DNA has been immobilized onto SAMs for biomaterial-directed gene delivery [36], [37], [38]. In the present work, we demonstrate SAM chemistry-dependent effects on retrovirus immobilization and gene delivery.
Section snippets
Cell culture
NIH3T3 murine fibroblasts (CRL-1658, American Type Culture Collection, Manassas, VA) were cultured in DMEM, 10% fetal bovine serum, 100 U/ml penicillin G sodium, and 100 μg/ml streptomycin sulfate in a humidified 5% CO2 atmosphere at 37 °C. Cell culture media and antibiotics were obtained from Invitrogen (Carlsbad, CA), fetal bovine serum was purchased from Hyclone (Logan, UT), and all other cell culture supplements and reagents were acquired from Sigma (St. Louis, MO).
Self-assembled monolayers
Alkanethiols 1-dodecanethiol
Results
To test the effects of surface chemistry on retrovirus immobilization to biomaterial surfaces, eGFP-encoding retroviral supernatants were incubated on CH3, COOH, or NH2 SAMs or tissue culture-treated polystyrene control (TC) for 16 h. Surfaces were then washed thoroughly with PBS and seeded with NIH3T3 murine fibroblasts. Expression of eGFP was measured by flow cytometry 72 h later as an indicator of retrovirus immobilization and biomaterial-mediated gene transfer (Fig. 1). In contrast to other
Discussion
Biomaterial-mediated gene delivery offers several advantages over conventional methods for genetic engineering of mammalian cells in vitro and in vivo. In particular, this strategy offers the potential to increase the efficiency of gene transfer by co-localizing the cells and viral particles and enhance the safety of gene therapy by avoiding off-target effects of the gene carrier on neighboring cells and tissues. This study characterizes a novel approach to retroviral gene transfer through
Conclusion
We describe a novel approach to biomaterial-mediated gene delivery via surface immobilization of retroviral particles onto alkanethiol self-assembled monolayers, which impart a permanent genetic modification in target cells. Biomaterial-assisted retroviral gene transfer exhibited strong dependence on the biomaterial surface chemistry as well as viral supernatant and cell culture conditions. This strategy is particularly efficient in engineering mammalian cells and compares favorably with other
Acknowledgments
The authors thank T.J. Murphy for generously providing the retroviral vector. This research was funded by the NIH (R01-EB003364), the Georgia Tech/Emory, NSF Engineering Research Center on Tissue Engineering (EEC-9731643), and a Medtronic Foundation Fellowship and NIH Biotechnology Training Grant (T32-GM08433).
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2014, BiomaterialsCitation Excerpt :Transport vectors have been categorized into two groups: viral and non-viral vectors [21]. In spite of the superior gene transfection efficiency [22], application of viral vectors was restricted because of their shortcomings of potential carcinogenicity, immunogenicity and complicated synthesis procedure [23]. The non-viral vectors, as an alternative safe and efficient gene carrier, arouse a wide concern [24].
Gene Delivery into Cells and Tissues
2013, Principles of Tissue Engineering: Fourth EditionA PLG/HAp composite scaffold for lentivirus delivery
2013, BiomaterialsCitation Excerpt :Reversible association of the lentivirus directly to the HAp embedded in the PLG/HAp scaffold allows for virus retention and preservation of activity. A number of approaches have been investigated for the immobilization of virus, which include vector biotinylation for binding to avidin-modified surface [5], immobilization of antibodies specific for the virus [22,23], phosphatidylserine (PS) functionalization for binding VSV-G pseudotyped lentivirus [6], and extracellular matrix protein [4] or self-assembled monolayer [24] sequestration. These approaches have identified the challenge of providing specific virus immobilization without reduction of activity due to virus destabilization [1].